The object of the present invention is a method for identifying one or a small number of molecules, especially in a dilution of xe2x89xa61 xcexcM, using laser excited fluorescence correlation spectroscopy, the use of said method in particular applications, as well as a device for performing the method according to the invention.
In recent years, analysis of biologically active molecules has been constantly improved in terms of specificity and sensitivity and supplemented by basically novel techniques. In this context, we may refer to cloning methods or the methods of enzyme-based amplification of genetic material to amplify single cells or molecules to such a number that they become apt to conventional analysis. In many cases, however, it would be more advantageous if analytic methods were sufficiently sensitive to qualitatively and quantitatively apply directly to single molecules or ensembles of a few molecules.
Electron microscopy, for instance, is a technique that can detect single molecules. Thus, attempts are made to sequence single DNA molecules by means of tunnel electron microscopy. This is very laborious, however.
Beyond the mere analysis of single molecules, information about state parameters of the molecules, such as their conformations and interactions with other molecules or molecular structures, are important in many fields.
Modern methods of evolutive biological engineering are concerned with highly complex collectives of molecules. Their object is to identify molecules having specific properties of interaction with target structures, that is to measure a particular fitness with respect to a desired function. Such fitness can be reduced to thermodynamic parameters such as binding constants or rate constants.
Sometimes it is less critical for the solution of particular problems to increase the sensitivity of an assay method, for instance when the molecule to be analyzed is present only in small concentrations. Rather, a very large number of samples which have to be analyzed more or less simultaneously must be coped with. If for instance 106 analyses have to be performed within a period of hours, it is obvious that only an analytic method can be considered where the samples can be measured and evaluated within a period of about 1 ms to 1 s as a maximum. The problem underlying the invention is, inter alia, to provide a method which, beyond the mere detection of single molecules, allows for informations about their specific interactions with other molecules or molecular structures to be obtained. Moreover, a very large number of samples is to be analyzed virtually simultaneously.
The method according to the invention is based on a luminescence detection and makes use of a technique which is known per se under the name of fluorescence correlation spectroscopy (FCS). Chromophorous molecular structures having fluorescence properties can be used to obtain information about the molecular environment of a chromophorous ligand. Rotational diffusion and translational diffusion of a luminophore may be measured as well as different paths of energy transfer to interacting molecules, chemical kinetics and the lifetime of excited states.
Based on physicochemical phenomena known per se, the method according to the invention provides novel solutions, making use of spectroscopic measuring parameters, for obtaining information from single molecules or a small number of molecules about the nature of said molecules as well as information about their fitness with respect to a particular interactional function or about the populations of different states of a luminophore which are defined with respect to one molecule.
To date, the method of fluorescence correlation spectroscopy as pursued by the groups of D. Magde (Elson, E. L. and Magde, D. (1974) Fluorescence correlation spectroscopy; Conceptional basis and theory; Biopolymers 13, 1-27) and R. Rigler (Ehrenberg, M. and Rigler, R. (1974) Rotational Brownian motion and fluorescence intensity fluctuations; Chem. Phys. 4, 390-401) for nearly twenty years could not be technically incorporated into a practicable analytical method without difficulty. It has not been possible to meet the above mentioned requirements with respect of measuring times and the light induced bleaching (photobleaching) of the dyes. Rigler et al. were able to determine rotational times of molecules. Magde et al. were able to determine some chemical reaction constants through fluctuation times.
The principle of measurement of FCS is to measure fluorophorous molecules in extremely diluted solutions (xe2x89xa610 nM) by exposing a relatively small volume element of the solution to the intense exciting light of a laser. Only the molecules having a corresponding exciting spectrum which are present in this same volume are excited by the light. Then, an image of the emitted fluorescence from this volume element can be formed on a photomultiplier with high sensitivity. If the solutions are diluted, significant variations of the concentration of the molecules present in the respective volume element will arise.
In particular, very diluted solutions will exhibit a Poisson distribution of the number of molecules which are simultaneously present within the volume element in a certain period of time. A molecule which has once diffused into the volume element will leave the volume element again within an average yet characteristic, for this type of molecule, period of time according to its characteristic diffusion rate (translational) and hence will not be observable any longer.
Now, if the luminescence of one and the same molecule can be excited many times during its average dwelling time within the respective observation element, many luminescence signals from this molecule can be detected. In other words, the probability that a molecule which has once diffused into the observation element can be excited once more before it will leave the volume element again is much greater in diluted solutions than would be true for a freshly entering molecule. Though this means that with a correspondingly great possibility the corresponding luminescence signal comes from one and the same molecule rather than from a molecule which has freshly entered the element. Hence, a correlation between the change with time of the incoming emission signals and the relative diffusion times of the molecular species involved can be established.
If the rotation of the polarization plane of exciting light and emitted light is measured as a further parameter, then the rotational diffusion coefficient of the molecules involved from which conclusions about molecular weight, shape parameters or the surrounding matrix can be obtained may also be determined.
It becomes evident that it is even possible to detect single molecules in diluted solutions by exciting one and the same molecule very often (several thousand times) and accumulating the corresponding luminescence signal from many single measurements.
The realization of this measuring principle in practice was impeded by many technical difficulties. Although modern laser technology was employed, the observation element was so large that biologically interesting molecules having low translational diffusion coefficients were present in the observation element during a period whose order of magnitude was about 50 ms. Such a period is significantly too large since it causes strong bleaching of the respective dye ligands employed serving as the luminophore. Frequent excitation increases the chemical reactivity of the luminophorous structure towards molecules of the environment, in particular oxygen, whereby the luminescence is altered or quenched. Of course, photobleaching also leads directly to false measuring data, since loss of luminescence (fluorescence) simulates the molecule""s leaving the measuring element and a distinction by standardizing the measuring method is hardly possible or can only be attained by unduly great technical expenditure.
To date, the wide practical realization of this measuring principle in a generally applicable method was hence restricted to within narrow limits which will be overcome, however, by the method according to the invention.
The critical breakthrough on the way to a routine method for the tasks of FCS as defined above according to the invention is achieved by the introduction of ultrasmall measuring volumes (preferably 10xe2x88x9214-10xe2x88x9217 l), with total volumes of the sample being in the xcexcl-range, the realization of which requires the simultaneous use, according to the invention, of particular elements of excitation optics, single photon detection and sample handling. The measuring volume of the device described in the experimental layouts is 2xc3x9710xe2x88x9216 l which is about 1000 times as small as the measuring volumes described in the literature. Hence, the illuminated area has an approximate dimension of 0.1 xcexcm2. Supposing that FCS provides correct data in particular for a concentration of chromophorous molecules of 0.1-10 molecules per measuring element volume, the working concentration will be about 10xe2x88x927-10xe2x88x929 M. Measurements with maximum detection efficiency and background discrimination can be technically realized by using confocal optics with high apertures in combination with single photon detection.
Binding constants can then be determined through translational diffusion if reaction time is slow compared with diffusion time. That means that a ligand which is to be observed does not change or changes but hardly its molecular structure during its entering into the measuring compartment and its leaving the same. Otherwise only a correlation is measured describing the mixed state. Here again, the importance of the method according to the invention using very small measuring volumes becomes evident, since the dwelling time of a molecule is about 1000 times shorter than with conventional compartments and hence opens up the usual dimensions of equilibrium constants and rate constants of specific biological recognition reactions, for example ligand/receptor interactions, for measurement.
According to the invention, the significance of the small volume elements for the performance of the methods according to the invention and the uses thereof has different, experimentally distinguishable aspects.
The significance of the small volume elements should be seen under the following aspects:
background scattered radiation, especially Raman radiation,
life of fluorescence dyes under light exposure,
short measuring times,
diffusion times of macromolecular complexes, viruses, cells,
integrity/preservation of the complexes not present in the volume element, and
life of complexes during measurement.